Use of p97 as an enzyme delivery system for the delivery of therapeutic lysosomal enzymes

ABSTRACT

The present invention provides for compositions and methods for treating, ameliorating or preventing a lysosomal storage disease by administering to a patient suffering from a lysosomal storage disease a P97 conjugated with an enzyme which is capable of transportation into the lysosomes of cells on either side of the blood brain barrier.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to and is a continuation of U.S. patentapplication Ser. No. 15/445,375, filed on Feb. 28, 2017, which is acontinuation of U.S. patent application Ser. No. 13/969,280, filed onAug. 16, 2013, which is a continuation of U.S. patent application Ser.No. 12/729,792, filed on Mar. 23, 2010, which issued as U.S. Pat. No.8,546,319, on Oct. 1, 2013, which is a continuation of U.S. patentapplication Ser. No. 10/501,028, filed on Mar. 14, 2004, which is a 371of international application number PCT/US03/00894, filed on Jan. 10,2003, which claims priority to U.S. provisional application No.60/347,758 filed on Jan. 11, 2002, the contents of each of which areincorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 3, 2020, isnamed 104321-600_SL.txt and is 8,991 bytes in size.

FIELD OF THE INVENTION

The present invention is related to the field of pharmaceutics andspecifically to treatment of diseases through the introduction of p97protein or polypeptide conjugated to a therapeutic or diagnostic agentto a subject. In particular, this invention relates to conjugates of p97and proteins deficient in a lysosomal storage disease and methods forthe treatment, amelioration or prevention of lysosomal storage diseasesby administration of the conjugates to subjects having such diseases.

BACKGROUND OF THE INVENTION

Lysosomal storage diseases (LSDs) result from the absence or reducedactivity of specific enzymes within the lysosomes of a cell. A largenumber of these enzymes have been identified and correlated with theirrelated diseases. Once the missing or deficient enzyme has beenidentified, treatment can be reduced to the problem of deliveringreplacement enzyme (drug) to a patient's affected tissues. Within cells,the effect of the missing enzyme can be seen as an accumulation ofundegraded “storage material” within the intracellular lysosome. Thisbuild-up causes lysosomes to swell and malfunction, resulting incellular and tissue damage. As lysosomal storage diseases typically havea genetic etiology, many tissues will lack the enzyme in question.However, different tissues suffer the absence of the same enzymedifferently. How adversely a tissue will be affected is determined, tosome extent, by the degree to which that tissue generates the substrateof the missing enzyme. The types of tissue most burdened by storage, inturn, dictate how the drug should be administered to the patient. Whileintravenous enzyme replacement therapy (ERT) is beneficial for LSDs(e.g. MPS I, MPS II), means for enhancing the delivery of thetherapeutic enzyme to the lysosome in such diseases would beadvantageous in terms of reduced cost and increased therapeuticefficacy.

In addition, the blood-brain barrier (BBB) blocks the free transfer ofmany agents from blood to brain. For this reason, LSDs that present withsignificant neurological affect (e.g. MPS III, MLD, GM1) are notexpected to be as responsive to intravenous ERT. For such diseases, amethod of delivering the enzyme across the BBB and into the lysosomes ofthe affected cells would be highly desirable.

In the early 1980's, melanotransferrin (MTf) or p97 was identified as anoncofetal antigen that was either not expressed, or only slightlyexpressed in normal tissues, but was found in much larger amounts inneoplastic cells (especially malignant melanoma cells) and fetal tissues(Woodbury, et al., P.N.A.S. USA, 77:2183-2187 (1980)). More recently,there have been additional reports of human MTf being identified innormal tissues, including sweat gland ducts, liver endothelial cells andthe endothelium and reactive microglia of the brain (Jefferies, et al.,Brain Res., 712:122-126 (1996); and Rothenberger, et al., Brain Res.,712:117-121 (1996)). Interestingly, normal serum contains very lowlevels of soluble circulating MTf, but increased soluble serum MTf hasbeen found in patients with advanced Alzheimer's Disease (Kennard, etal., Nat. Med., 2:1230-1235 (1996); U.S. Pat. No. 5,981,194).

The biochemical role and metabolism of MTf has proven difficult toelucidate. Based on appearances, MTf is deceptively similar totransferrin (Tf) and lactotransferrin (lactoferrin or Lf). In humans,these proteins share a 37-39% amino acid sequence homology. Inparticular, each of these proteins reversibly binds iron, and theirN-terminal iron binding domains are quite similar (Baker, et al., TIBS,12:350-353 (1987)).

However, functional parallels between these proteins have not beenconfirmed. For one thing, unlike Tf and Lf, MTf exists in both amembrane bound form and a serum soluble form. Further, in contrast to Tfand Lf, no cellular receptor for MTf has been identified. Serum solubleTf is known to be taken into cells in an energy-dependent processmediated by the transferrin receptor (Tf-R) (Cook, et al., Annu. Rev.Med., 44:63-74) (1993)). Lf internalization is also likely to bemediated by a receptor mediated process (Fillebeen, et al., J. Biol.Chem., 274(11):701-7017 (1999)). Two known receptors for Lf are LRP-1and RAGE, although others may exist (Melinger, et al., FEBS Letters,360:70-74 (1995); Schmidt, J. Biol. Chem., 269(13):9882-9888 (1994).

With respect to the central nervous system (e.g., brain, spinal cord),there are at least three ways to enhance delivery: direct injection,permeabilization of the BBB, and modification of the drug. Directinjection involves injection of drug into brain tissue, bypassing thevasculature completely. This method suffers primarily from the risk ofcomplications (infection, tissue damage) incurred by intra-cranialinjections. This risk is compounded when considered in the context of aregular treatment regimen applied over the course of to the patient'slife. It is also difficult, using a limited number of single siteinjections, to match the penetration that blood vessels (and hence,potentially, drug) have throughout the brain.

The second method entails non-specifically compromising the BBB withconcomitant injection of intravenous drug. Permeabilization of the BBBis accomplished chemically. This method suffers from a lack ofspecificity. All those components in the blood that are necessarilyexcluded by the BBB will enter the brain along with the drug. The brainis left vulnerable under these conditions and damage would beanticipated over the course of a life-long regimen of treatment.

The third means of increasing brain availability of blood borne drugentails specific functionalization of the drug with moieties thatfacilitate transport through an uncompromised BBB. This method has theadvantages of specific BBB infiltration and convenient intravenousadministration. A method of increasing the ability of a therapeuticagent to cross the blood brain barrier is taught in U.S. Pat. No.6,455,494, incorporated herein by reference its entirety, whichdiscloses the use of p97 as a carrier for delivering a therapeutic drugacross the blood brain barrier.

p97 (melanotransferrin) is a naturally occurring human protein. p97 wasdiscovered and characterized as a cell surface marker for human melanoma(melanoma-associated antigen), but has more recently been found in othertumor types, as well as in normal human brain and liver tissue, in traceamounts in other body tissues and in serum. The role of p97 in the bodyis unknown, but based on its structure and binding properties, it isthought to be involved in the transport of metal ions (e.g. iron) intocells. Jefferies, et al. have been working with p97 since 1992 (U.S.Pat. No. 5,981,194). These investigations have focused on p97 as adiagnostic marker for Alzheimer's disease (AD). Synapse has developed ablood (serum) test for AD that is based on the finding that the p97serum level increases with the progression of the disease. During thedevelopment of this test, it was discovered that p97 is activelytransported from the blood into the brain tissue of normal individuals.This discovery was the impetus for the development of p97 as a potentialtransport system to deliver molecules from the blood, across the BBB toreach brain interstitial fluids.

The key event for the successful delivery of therapeutic agents intobrain is the transport of these large molecules across the tight networkof capillary endothelial cells that comprise the BBB. During the lastfew years, it has been demonstrated both in vitro and in animal modelsthat small synthetic molecules, large glycoprotein enzymes, and largeinorganic particles (5 nm colloidal gold particles) chemically linked top97, can be transported across the BBB to brain cells. Such transport oflarge molecules across the BBB involves a process known as transcytosis.This is a mechanism whereby molecules are picked up from the blood andtransported through the capillary cells of the otherwise intact BBB tothe brain tissue.

Transcytosis pathways are distinct from other vesicular traffic withinthe capillary endothelial cell and transit occurs without alteration ofthe transported materials. Transcytosis is a cell-type specific processmediated by receptors on the BBB endothelial surface. The transport ofp97-conjugates (i.e., p97 chemically linked to macromolecules) acrossthe BBB occurs by transcytosis. p97 conjugated to the enzyme horseradishperoxidase (HRP) (an example of an enzyme “payload”) can be transportedacross the BBB.

For an effective treatment of LSDs, a therapeutic agent (e.g., thedeficient enzyme, or another enzyme or protein having a desiredtherapeutic or missing enzyme activity) must be taken up by the affectedcells and routed to the lysosome where it acts upon the excessive orharmful amount of storage material residing therein. Applicants providebelow compositions and methods for treating LSDs involving the use ofp97 proteins to target delivery of therapeutic agents, includingproteins or enzymes deficient in LSDs, to the lysosomes of cells.

BRIEF SUMMARY OF THE INVENTION

The present invention is related to the discovery that conjugates of p97and therapeutic agent, in which the agent is covalently linked to p97,or to a fragment or portion thereof, are excellent vehicles for theenhanced delivery of the agent to lysosomes of cells within and withoutthe CNS. In a first aspect, therefore the invention provides a method ofdelivering a therapeutic agent to the lysosome of a cell. In a secondaspect the invention provides a method of treating a lysosomal storagedisease in a patient by administering a p97 molecule covalently linkedto a therapeutic agent which is a protein or enzyme deficient in thelysosomes of a subject having such a disease (e.g., enzyme replacementtherapy). Such p97-agent conjugates are particularly useful, forexample, in the treatment of lysosomal storage diseases such as MPS I,MPS II, MPS III A, MPS III B, Metachromatic Leukodystrophy, Gaucher,Krabbe, Pompe, CLN2, Niemann-Pick and Tay-Sachs disease wherein alysosomal protein deficiency contributes to the disease state. In athird aspect, the invention provides pharmaceutical compositionscomprising a p97 molecule covalently linked to a protein or enzymedeficient in a lysosomal storage disease. In a fourth aspect, theinvention provides methods for identifying p97 conjugates which areuseful in delivering an agent to a lysosome.

In some embodiments, the methods of the invention can be used to treatsuch lysosomal storage diseases as Aspartylglucosaminuria, Cholesterolester storage disease/Wolman disease, Cystinosis, Danon disease, Fabrydisease, Farber Lipogranulomatosis/Farber disease, Fucosidosis,Galactosialidosis types I/II, Gaucher disease types I/IIIII Gaucherdisease, Globoid cell leucodystrophy/Krabbe disease, Glycogen storagedisease II/Pompe disease, GM1-Gangliosidosis types I/II/III,GM2-Gangliosidosis type I/Tay Sachs disease, GM2-Gangliosidosis type IISandhoff disease, GM2-Gangliosidosis, α-Mannosidosis types I/II,β-Mannosidosis, Metachromatic leucodystrophy, Mucolipidosis typeI/Sialidosis types I/II Mucolipidosis types II/III I-cell disease,Mucolipidosis type IIIC pseudo-Hurler polydystrophy,Mucopolysaccharidosis type I, Mucopolysaccharidosis type II Huntersyndrome, Mucopolysaccharidosis type IIIA Sanfilippo syndrome,Mucopolysaccharidosis type IIIB Sanfilippo syndrome,Mucopolysaccharidosis type IIIC Sanfilippo syndrome,Mucopolysaccharidosis type IIID Sanfilippo syndrome,Mucopolysaccharidosis type IVA Morquio syndrome, Mucopolysaccharidosistype IVB Morquio syndrome, Mucopolysaccharidosis type VI,Mucopolysaccharidosis type VII Sly syndrome, Mucopolysaccharidosis typeIX, Multiple sulphatase deficiency, Neuronal Ceroid Lipofuscinosis, CLN1Batten disease, Neuronal Ceroid Lipofuscinosis, CLN2 Batten disease,Niemann-Pick disease types AB Niemann-Pick disease, Niemann-Pick diseasetype C1 Niemann-Pick disease, Niemann-Pick disease type C2 Niemann-Pickdisease, Pycnodysostosis, Schindler disease types I/II Schindlerdisease, and Sialic acid storage disease.

In some embodiments, the p97 conjugates compositions comprise from about1 to about 5 molecules of the agent of interest linked to a single p97molecule. In some embodiments, more that one agent of interest may belinked to a single p97 molecule. Selective biodistribution of p97-agentscan enhance the selective targeting of p97-linked agents to specificorgans.

In addition, the present invention provides screening assays foridentifying p97-agent conjugates that can prevent, ameliorate, or treata lysosomal storage disease by contacting a cell containing a lysosomewith the conjugate and determining whether the conjugate delivers theagent to the lysosome. The delivery can be assessed by labeling theconjugate and then monitoring or detecting the location of the label inthe cell or by determining the effect of the conjugate on the amount ofthe storage material found in the lysosome. In a preferred embodiment,the agent is a protein or enzyme deficient in the lysosomal storagedisease. In another embodiment, the cell is deficient in the agentconjugated to the p97 molecule.

In some embodiments, the method treats lysosomal storage diseaseswherein the tissues to be treated are isolated from the circulatorysystem by the blood brain barrier (e.g., the brain). In one embodiment,the present invention provides a method for delivering a compound ofinterest through the blood-brain barrier of a subject into a lysosome ofa cell of the subject comprising: administering a conjugated agent tothe subject, wherein the conjugated agent comprises a melanotransferrinlinked to the agent via a linker; whereby the conjugated agent passesthrough the blood-brain barrier of the subject, and whereby the agententers into a lysosome of a cell of the subject.

In another embodiment, the present invention provides for a method fortreating, ameliorating, or preventing a lysosomal storage disease of asubject comprising: administering a conjugated agent to a subject,wherein the conjugated agent comprises a melanotransferrin linked to aan agent of interest via a linker; whereby the agent enters into alysosome of a peripheral cell (e.g., non-CNS cell) of the subject.

In still another embodiment, the present invention provides for a methodof enzyme replacement therapy comprising: administering a conjugatedagent to a subject in need of the enzyme replacement therapy, whereinthe conjugated agent comprises a melanotransferrin linked to an enzymevia a linker, wherein the cells of the patient have lysosomes whichcontain insufficient amounts of the enzyme to prevent or reduce damageto the cells; whereby the conjugated agent passes through theblood-brain barrier of the subject, and whereby sufficient amounts ofthe enzyme enter the lysosomes to prevent or reduce damage to the cells.

In even another embodiment, the present invention provides a method fortreating a patient suffering from a lysosomal storage disease resultingfrom the absence of an enzyme within the lysosomes of a cell found inthe brain comprising: administering to the patient a conjugated agent,wherein the conjugated agent comprises p97 linked to the enzyme via alinker; whereby the conjugated agent passes through the blood-brainbarrier of the patient, and whereby sufficient amounts of the enzymeenter the lysosomes to prevent or reduce damage to the cells.

In yet even another embodiment, the present invention provides for amethod for identifying an agent that can prevent, ameliorate or treat alysosomal storage disease, comprising: administering a p97-conjugatedagent to a cell, wherein absence of the enzyme causes the lysosomalstorage disease; and determining whether the agent reduces damage to thecell compared to damage to the cell if the conjugated agent was notadministered to the cell. In certain embodiments, the method is a highthroughput assay.

In a further embodiment, the present invention provides for a novelcomposition comprising a conjugated agent, wherein the conjugated agentcomprises a p97 molecule linked to an enzyme via a linker, wherein theenzyme is an enzyme, such as those set forth herein, found in thelysosomes of cells contained within the BBB. The composition can furthercomprise a suitable pharmaceutical carrier.

Further, the present invention provides a lysosome comprising theconjugated agent. The present invention also provides a cell comprisinga lysosome comprising the conjugated agent. Preferably, the cell is acell found surrounded by a blood-brain barrier. More preferably, thecell is a neuron or a brain cell.

In some embodiments, the p97-agent conjugate comprises any one of thefollowing proteins as the active agent covalently linked to a p97molecule: aspartylglucosaminidase, acid lipase, cysteine transporter,Lamp-2, α-galactosidase A, acid ceramidase, α-L-fucosidase,β-hexosaminidase A, GM2-activator deficiency, α-D-mannosidase,β-D-mannosidase, arylsulphatase A, saposin B, neuraminidase,α-N-acetylglucosaminidase phosphotransferase, phosphotransferaseγ-subunit, L-iduronidase, iduronate-2-sulphatase, heparan-N-sulphatase,α-N-acetylglucosaminidase, acetylCoA:N-acetyltransferase,N-acetylglucosamine 6-sulphatase, galactose 6-sulphatase,β-galactosidase, N-acetylgalactosamine 4-sulphatase,hyaluronoglucosaminidase, multiple sulphatases, palmitoyl proteinthioesterase, tripeptidyl peptidase I, acid sphingomyelinase,cholesterol trafficking, cathepsin K, α-galactosidase B, sialic acidtransporter. In some embodiments, the agent is a protein of human ormammalian sequence, origin or derivation. The p97 protein or fragmentcan also be of human or mammalian sequence, origin or derivation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict human hNT neurons (derived from humanteratocarcinoma) stained with anti-Cathepsin L and the L235 monoclonalantibody to p97 (see, Example 1 for details).

FIGS. 2A-2C depict live neurons fed with AlexaFluor 594-p97 andlysosensor green (see, Example 2 for details).

FIG. 3 shows the general construct of a p97-conjugate covalentlyconjugated to an agent.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention generally provides methods and compositions forenhanced delivery of the lysosomal storage disease related agents to thelysosomes of cells affected by a lysosomal storage disease. Theinvention relates to the surprising discovery that MTf or p97 proteinsand fragments thereof not only undergo transcytosis across the bloodbrain barrier but also are transported into lysosomes. As a result, p97molecules which are demonstrated vehicles for delivering agents acrossthe blood brain barrier are also surprisingly particularly useful alsoas a means of delivering deficient proteins or enzymes to lysosomes forthe treatment of lysosomal storage diseases resulting from suchdeficiencies.

The conjugate agents of the present invention offer many importantadvantages in the treatment of lysosomal storage diseases. The MTf orp97 is a natural compound usually found in human cells at differentlevels. Because p97 is a natural protein of humans, it is unlikely toresult in immunological hyper-responsiveness, as it is frequently thecase with the use of Mab therapies making them is refractory for use inrepeated injections.

In terms of drug delivery, the p97 system can cross the BBB quickly(within an hour), and is metabolized within 12 hours, thus it isefficiently eliminated from tissues. These features provide the chanceof repetitive injections without saturation of the receptors. Moreover,p97 does not compete with endogenous transferrin whose amount isestimated to be 10,000 times higher than serum p97. Thus transferringwill not compete with the method of delivering therapeutic agents bycovalently or otherwise linking them to p97. The protein p97 showslittle toxicity if any. The p97 protein is biodegradable and will notcirculate for long periods of time. The protein has a demonstratedcapacity to cross the BBB and due to its affinity for the endothelialcells lining the vascular bed of the brain, p97 vector is particularlyuseful for delivering a conjugated or otherwise bound agent to lysosomeslocated in within the brain tissue.

II. Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Each publication, patentapplication, patent, and other reference cited herein is incorporated byreference in its entirety to the extent that it is not inconsistent withthe present disclosure.

It is noted here that as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise.

MTf or p97 includes membrane bound p97 (i.e., p97 attached to a GPIanchor or some other anchor), secreted p97, soluble p97, cleaved p97,analogs of p97 which are functional equivalents of p97 (having generallygreater than 40%, 60%, 80%, 90% or 95% homology of their correspondingamino acid sequences and including allelic variants of p97), human,mouse, chicken and/or rabbit p97, and derivatives, portions, orfragments thereof. The p97 can be in the form of acidic or basic salts,or in its neutral form. In addition, individual amino acid residues canbe modified, such as by oxidation or reduction. Moreover, varioussubstitutions, deletions, or additions can be made to the amino acid ornucleic acid sequences, the net effect of which is to retain or improveupon the desired biological activity of p97. Due to code degeneracy, forexample, there may be considerable variation in nucleotide sequencesencoding the same amino acid sequence. Further characterization of p97,including the complete amino acid sequence of p97, is found in U.S. Pat.No. 5,981,194.

A p97 fragment as used herein includes any portion of p97 or itsbiologically equivalent analogs that contains a sufficient portion ofp97 to enable it to bind to the MTf or the LRP1 or LRP1B receptor and tobe transported across the blood-barrier; or that otherwise retains orimproves upon the desired biological activities of p97 in thetranscytosis and/or delivery of an agent to the lysosome.

Additional teachings regarding the making and using of p97 as diagnosticand therapeutic agents is found in International Application No.PCT/CA93/00272 and U.S. Pat. No. 5,981,194, the disclosures of which areincorporated by reference in their entirety.

“Modulate,” as used herein, refers to the ability to alter, by increaseor decrease (e.g., to act as an antagonist or agonist).

“Melanotransferrin receptor” (“MTf-R”), as used herein, refers to anybiological system that specifically or preferentially binds MTf. Thisterm is intended to include those receptors which competitively bind Lfand/or β-amyloid protein, but excludes those receptors which arespecific for Tf such as the transferrin receptor (Tf-R) (which isdescribed at OMIM #*190010, and which is also known as TFR, TRFR andCD71). A receptor known to specifically or preferentially bind Lf isherein called a “lactotransferrin receptor” (Lf-R). Known Lf-Rs include,but are not limited to, the LDL-related receptors. A known LDL-relatedreceptor is lipoprotein receptor-related protein/alpha2- macroglobulinreceptor (“LRP1”). The term MTf-R specifically includes other receptorsfound on endothelial cells that specifically bind both MTf and Lf, butnot Tf. In a preferred embodiment, the MTf-R is the LRP1. In a morepreferred embodiment, the MTf-R is LRP1B.

Members of the low density lipoprotein (LDL) receptor family includeLDL-R (132 kDa); LRP/LRP1 and LRP1B (600 kDa); Megalin ((LRP2), 600kDa); VLDL-R (130 kDa); ER-2 (LRP-8, 130 kDa); Mosaic LDL-R (LR11, 250KDa); and other members such as LRP3, LRP6, and LRP-7. Characteristicfeatures of the LDL-R family include cell-surface expression;extracellular ligand binding domain repeats (DxSDE) (SEQ ID NO:9);requirement of Ca++ for ligand binding; recognition of RAP and ApoE; EGFprecursor homology domain repeats (YWTD) (SEQ ID NO: 10); singlemembrane spanning region; internalization signal in the cytoplasmicdomain (FDNPXY) (SEQ ID NO: 11); and receptor mediated endocytosis ofvarious ligands.

LRP refers to the low density lipoprotein receptor related protein andmembers of this receptor family. LRP is a large protein of 4525 aminoacids (600 kDa) which is cleaved by furin to produce two subunits of515-(α) and 85-(β) kDa that remain non-covalently bound. LRP is mainlyexpressed in the liver, kidney, neuron, CNS, BBB, SMC and variouscultured cells.

LRP ligands refer to a number of molecules that are known to bind LRP.These molecules include, for instance, lactoferrin, RAP, lipoproteinlipase, ApoE, Factor VIII, β-amyloid precursor, α2-macroglobulin,thrombospondin 2 MMP-2, MPP-9-TIMP-1; uPA:PAI-I:uPAR; andtPA:PAI-1:uPAR.

LRP 1B is a recently discovered member of the low density lipoproteinreceptor family. 600 kDa multifunctional cell surface receptor. See Liuet al., J. Biol. Chem. 276 (31):28889-28896 (2001). See also Liu et al.,Genomics 69, 271-274 (2000); and Liu et al., Cancer Res. 60, 1961-1967(2000). This receptor is more closely related to LRP than megalin andshares a 59% homology at cDNA level and a 52% homology at predictedamino acid level. the LRP 1B gene is expressed in the brain, thyroid andsalivary gland. Known ligands for LRP 1B include RAP, tPA, PAI-1.

Mouse LRP1B is accessible through GenBank Accession Nos. XM 143023 XM130241. Human LRP1B is accessible through GenBank Accession Nos. XM015452.

“Lipoprotein receptor-related protein/alpha2-macroglobulin receptor”(“LRP1”), as used herein, refers to a multifunctional receptor. It isbelieved that the clustering of cysteine-rich type. A binding repeats,resembling those found in the LDL receptor, is the molecular principlefor the ability to bind a variety of ligands that were previouslythought to be unrelated: activated alpha-2-macroglobulin, apolipoproteinE, lipoprotein lipase, plasminogen activators and complexes with theirinhibitor (PA and PA/PAI-1), lipoprotein(a), pseudomonas exotoxin A,human rhinovirus, Lf and the so-called receptor associated protein(RAP). See, Meilinger, et al., FEBS Let., 360:70-74 (1995). LRPI isaccessible through GenBank Accession No.: X 13916 and Swiss-Prot PrimaryAccession No.: Q07954. Alternative names for the LRP1 gene/proteininclude: Low-density lipoprotein receptor-related protein 1 [precursor],LRP, Alpha-2-macroglobulin receptor, A2MR, Apolipoprotein E receptor,APOER, CD91, LRP1 or A2MR.

A “p97-conjugate” refers to a composition comprising p97, or a fragmentthereof, covalently conjugated to an agent. The conjugation may bedirect or indirect (i.e., through an extended linker) so long as it is achemical conjugation. The general construct of the composition of theinvention is shown in FIG. 3.

The agent may serve a therapeutic or prophylactic purpose in thetreatment of a lysosomal storage disease in mammals, and particularly,humans.

“Increasing relative delivery” as used herein refers to the effectwhereby the accumulation at the lysosome of a p97-conjugated agent isincreased relative to the accumulation of the original agent.

“Therapeutic index” refers to the dose range (amount and/or timing)above the minimum therapeutic amount and below an unacceptably toxicamount.

“Equivalent dose” refers to a dose which contains the same amount ofactive agent.

The p97-agent conjugate can comprise one or more agent moieties (e.g., 1to 10 or 1 to 4 or 2 to 3 moieties) linked to p97. For example,conjugation reactions may conjugate from 1 to 4 or more molecules ofalpha-L-iduronidase to a single p97 molecule. These formulations can beemployed as mixtures, or they may be purified into specific p97:agent(mol:mol) formulations. Those skilled in the art are able to determinewhich format and which mol:mol ratio is preferred. Further, mixtures ofagents may be linked to p97 to facilitate a more complete degradation ofthe stored substrates. These p97-agents may consist of a range ofmol:mol ratios. These, too, may be separated into purified mixtures orthey may be employed in aggregate.

The p97 conjugated agents can enter or be transported into or end upresiding in the lysosomes of a cell within or without the CNS. The rateof passage of the conjugated agent can be modulated by any compound orprotein that can modulate a MTf receptor activity. Methods foridentifying or determining such a modulator are disclosed in U.S.Provisional Patent Application Ser. No. 60/308,002 and U.S. patentapplication Ser. No. 10/206,448, filed on Jul. 25, 2002, the disclosuresof which are incorporated by reference in their entirety. The cell canbe from any tissue or organ system affected by the lysosomal storagedisease. The cell can be, for instance, an endothelial, epithelial,muscle, heart, bone, lung, fat, kidney, or liver cell. In someembodiments, the cell is preferably a cell found within the BBB. In someembodiments, the cell is a neuron or a brain cell. In other embodiments,the cell is a cell of the periphery or one which is not isolated fromthe general circulation by an endothelium such as that of the BBB.

The agent can be a protein or enzyme or any fragment of such that stillretains some, substantially all, or all of the activity of the enzyme.In some embodiments, the protein or enzyme is one that, if not expressedor produced or if substantially reduced in expression or production,would give rise to a lysosomal storage disease. Preferably, the proteinor enzyme is derived or obtained from a human or mouse. In someembodiments, in the treatment of human LSDs, the p97-agent conjugatecomprises a protein or enzyme that is deficient in the lysosomal storageenzyme deficient in subject or patient to be treated. Such enzymes,include for example, α-L-iduronidase, iduronate-2-sulfatase, heparanN-sulfatase, α-N-acetylglucosaminidase, Arylsulfatase A,Galactosylceramidase, acid-alpha-glucosidase, thioesterase,hexosaminidase A, Acid Spingomyelinase, α-galactosidase, or any otherlysosomal storage enzyme. A table of lysosomal storage diseases and theproteins deficient therein follows:

Lysosomal Storage Disease Protein deficiency Mucopolysaccharidosis typeI L-Iduronidase Mucopolysaccharidosis type II Hunter syndromeIduronate-2-sulphatase Mucopolysaccharidosis type IIIA Sanfilipposyndrome Heparan-N-sulphatase Mucopolysaccharidosis type IIIB Sanfilipposyndrome α-N-Acetylglucosaminidase Mucopolysaccharidosis type IIICSanfilippo syndrome AcetylCoA:N-acetyltransferase Mucopolysaccharidosistype IIID Sanfilippo syndrome N-Acetylglucosamine 6-sulphataseMucopolysaccharidosis type IVA Morquio syndrome Galactose 6-sulphataseMucopolysaccharidosis type IVB Morquio syndrome β-GalactosidaseMucopolysaccharidosis type VI N-Acetylgalactosamine 4-sulphataseMucopolysaccharidosis type VII Sly syndrome β-GlucuronidaseMucopolysaccharidosis type IX hyaluronoglucosaminidaseAspartylglucosaminuria Aspartylglucosaminidase Cholesterol ester storagedisease/Wolman disease Acid lipase Cystinosis Cystine transporter Danondisease Lamp-2 Fabry disease α-Galactosidase A FarberLipogranulomatosis/Farber disease Acid ceramidase Fucosidosisα-L-Fucosidase Galactosialidosis types I/II Protective protein Gaucherdisease types I/IIIII Gaucher disease Glucocerebrosidase (β-glucosidase)Globoid cell leucodystrophy/Krabbe disease Galactocerebrosidase Glycogenstorage disease II/Pompe disease α-Glucosidase GM1-Gangliosidosis typesI/II/III β-Galactosidase GM2-Gangliosidosis type I/Tay Sachs diseaseβ-Hexosaminidase A GM2-Gangliosidosis type II Sandhoff diseaseβ-Hexosaminidase A GM2-Gangliosidosis GM2-activator deficiencyα-Mannosidosis types I/II α-D-Mannosidase β-Mannosidosis β-D-MannosidaseMetachromatic leucodystrophy Arylsulphatase A Metachromaticleucodystrophy Saposin B Mucolipidosis type I/Sialidosis types I/IINeuraminidase Mucolipidosis types II/III I-cell diseasePhosphotransferase Mucolipidosis type IIIC pseudo-Hurler polydystrophyPhosphotransferase γ-subunit Multiple sulphatase deficiency Multiplesulphatases Neuronal Ceroid Lipofuscinosis, CLN1 Batten diseasePalmitoyl protein thioesterase Neuronal Ceroid Lipofuscinosis, CLN2Batten disease Tripeptidyl peptidase I Niemann-Pick disease types A/BNiemann-Pick disease Acid sphingomyelinase Niemann-Pick disease type C1Niemann-Pick disease Cholesterol trafficking Niemann-Pick disease typeC2 Niemann-Pick disease Cholesterol trafficking PycnodysostosisCathepsin K Schindler disease types I/II Schindler diseaseα-Galactosidase B Sialic acid storage disease Sialic acid transporter

The melanotransferrin or p97 and the agent are conjugated directly orindirectly to each other (i.e., through an extended linker). The linkercan comprise a covalent bond or a peptide of virtually any amino acidsequence or any molecule capable of conjugating melanotransferrin or p97and the agent. If the linker is a covalent bond or a peptide, then theentire conjugate can be a fusion protein. Such fusion proteins may beproduced by recombinant genetic engineering methods known to one ofordinary skill in the art.

The p97-enzyme conjugate according to the invention may be modified asdesired to enhance its stability or pharmacokinetic properties (e.g.,PEGylation).

III. Compositions and Preparation Thereof

In general, p97-conjugates may be prepared using techniques well knownin the art. There are numerous approaches for the conjugation orchemical crosslinking of agents to a polypeptide such as p97, and oneskilled in the art can determine which method is most appropriate forconjugating a particular agent. The method employed must be capable ofjoining the agent with p97 without interfering with the ability of p97to bind to its receptor, preferably without influencing thebiodistribution of the p97-agent compared to p97 alone, and/or withoutsignificantly altering the desired activity of the agent (be ittherapeutic or prophylactic or the like) once delivered. Preferredmethods of conjugating p97 to various agents are described in theexamples section, below. A particularly preferred method for linkingcomplex molecules to p97 is the SATA/sulfo-SMCC cross-linking reaction(Pierce (Rockford, Ill.)).

Methods of cross linking proteins and peptides are well known to thoseof skill in the art. Several hundred crosslinkers are available forconjugating a compound of interest with p97 or with a substance whichbinds p97 (see, e.g., Chemistry of Protein Conjugation and Crosslinking,Shans Wong, CRC Press, Ann Arbor (1991) and U.S. Pat. No. 5,981,194 andPCT Patent Publication Nos. WO 02/13843 and WO 01/59459 which areincorporated herein by reference in their entirety). Many reagents andcross-linkers can be used to prepare conjugates of an active agent and ap97 molecule. See, for instance, Hermanson, G T et al. BioconjugateTechniques, Academic Press, (1996). The crosslinker is generally chosenbased on the reactive functional groups available or inserted on thetherapeutic agent. In addition, if there are no reactive groups, aphotoactivatible crosslinker can be used. In certain instances, it maybe desirable to include a spacer between p97 and the agent. In oneembodiment, p97 and the protein therapeutic agents may be conjugated bythe introduction of a sulfhydryl group on p97 and by the introduction ofa crosslinker containing a reactive thiol group on to the proteincompound through carboxyl groups (Wawizynczak and Thorpe inImmunoconjugates: Antibody Conjugates in Radioimaging and Therapy ofCancer, Vogel (Ed.) Oxford University Press, pp. 28-55 (1987); and Blairand Ghose (1983) J. Immunol. Methods 59:129). In some embodiments, thelinker is vulnerable to hydrolysis at the acidic pH of the lysosome soas to free the agent from the p97 and/or linker.

In some embodiments of the present invention, the p97-agent conjugate isa p97-fusion protein. Fusion proteins may be prepared using standardtechniques known in the art. Typically, a DNA molecule encoding p97 or aportion thereof is linked to a DNA molecule encoding the proteincompound. The chimeric DNA construct, along with suitable regulatoryelements can be cloned into an expression vector and expressed in asuitable host. The resultant fusion proteins contain p97 or a portionthereof used to the selected protein compound.

When a linker is used, the linker is preferably an organic moietyconstructed to contain an alkyl, aryl and/or amino acid backbone, andcontaining an amide, ether, ester, hydrazone, disulphide linkage or anycombination thereof. Linkages containing amino acid, ether and amidebound components are stable under conditions of physiological pH,normally 7.4 in serum. Preferred linkages are those containing esters orhydrazones that are stable at serum pH, but that hydrolyze to releasethe drug when exposed to lysosomal pH. Disulphide linkages are preferredbecause they are sensitive to reductive cleavage. In addition, aminoacid linkers may be designed to be sensitive to cleavage by specificenzymes in the desired target organ or more preferably, the lysosomeitself. Exemplary linkers are described in Blattler et al. (1985)Biochem. 24:1517-1524; King et al. (1986) Biochem. 25:5774-5779;Srinivasachar and Nevill (1989) Biochem. 28:2501-2509.

In some embodiments, the linker is a polyethylene glycol orpolypropylene glycol. In other embodiments, the linker is from 4 to 20atoms long. In other embodiments, the linker is from 1 to 30 atoms longwith carbon chain atoms which may be substituted by heteroatomsindependently selected from the group consisting of O, N, or S. In someembodiments, from 1-4 or from 5 to 15 of the C atoms are substitutedwith a heteroatom independently selected from O, N, S. In otherembodiments, the linker contains a moiety subject to hydrolysis upondelivery to the lysosomal environment (e.g., susceptible to hydrolysisat the lysosomal pH or upon contact to a lysosomal enzyme). In someembodiments, the linker group is preferably hydrophilic to enhance thesolubility of the conjugate in body fluids. In some embodiments, thelinker contains or is attached to the p97 molecule or the protein agentby a functional group subject to attack by other lysosomal enzymes(e.g., enzymes not deficient in the target lysosome or a lysosomalenzyme not conjugated to the p97 carrier). In some embodiments, the p97and agent are joined by a linker comprising amino acids or peptides,lipids, or sugar residues. In some embodiments, the p97 and agent arejoined at groups introduced synthetically or by posttranslationalmodifications.

In some embodiments, agent-linker intermediates are similar to what hasbeen described previously, but comprise, for example, either an activeester that can react with free amine groups on p97 or a maleimide thatcan react with the free thiols created on p97 via a SATA reaction orthrough other groups where persons skilled in the art can attach them top97.

A. Preparation of p97

The p97 peptide or molecule for use in the methods and compositions ofthe present invention may be obtained, isolated or prepared from avariety of sources.

In one aspect, standard recombinant DNA techniques may be used toprepare p97 or derivatives thereof. Within one embodiment, DNA encodingp97 may be obtained by polymerase chain reaction (PCR) amplification ofthe p97 sequence (see, generally, U.S. Pat. Nos. 4,683,202; 4,683,195;and 4,800,159; see, also, PCR Technology: Principles and Applicationsfor DNA Amplification, Erlich (ed.), Stockton Press (1989)). Briefly,double-stranded DNA from cells which express p97 (e.g., SK-MEL-28 cells)is denatured by heating in the presence of heat stable Taq polymerase,sequence specific DNA primers such as 5′ GCGGACTTCCTCGG 3′ (SEQ ID NO:8)and 5′ TCGCGAGCTTCCT 3′ (SEQ ID NO:2), ATP, CTP, GTP and TTP.Double-stranded DNA is produced when the synthesis is complete. Thiscycle may be repeated many times, resulting in a factorial amplificationof p97 DNA. The amplified p97 DNA may then be readily inserted into anexpression vector as described below.

Alternatively, DNA encoding p97 may be isolated using the cloningtechniques described by Brown et al. in the UK Patent Application No. GB2188 637. Clones which contain sequences encoding p97 cDNA have beendeposited with the American Type Culture Collection (ATCC) under depositnumbers CRL 8985 (PMTp97b) and CRL 9304 (pSVp97a).

Within one embodiment of the present invention, truncated derivatives ofp97 are provided. For example, site-directed mutagenesis may beperformed with the oligonucleotide WJ31 5′CTCAGAGGGCCGCTGCGCCC-3′ (SEQID NO:3) in order to delete the C-terminal hydrophobic domain beyondnucleotide 2219, or with the oligonucleotide WJ32 5′ CCA GCG CAGCTAGCGGGGGCAG 3′ (SEQ ID NO:4) in order to introduce an Nhe I site and aSTOP codon in the region of nucleotides 1146-1166, and thereby alsoconstructing a truncated form of p97 comprising only the N-terminaldomain. Similarly, mutagenesis may also be performed on p97 such thatonly the C-terminal domain is expressed. Within one embodiment, Xhosites are inserted by mutagenesis with the oligonucleotide WJ5′-ACACCAGCGCAGCTCGAGGGGCAGCCG 3′ (SEQ ID NO:5) into both the N-terminaland C-terminal domains, allowing subsequent deletion of the N-terminaldomain. Various other restriction enzymes, including for example, EcoRI, may also be utilized in the context of the present invention inorder to construct deletion or truncation derivatives of p97.

Mutations may be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling the ligation of the mutated fragments to fragments of thenative sequence. Following ligation, the resulting reconstructedsequence encodes a derivative having the desired amino acid insertion,substitution, or deletion. Alternatively, as noted aboveoligonucleotide-directed site-specific mutagenesis procedures may beemployed to obtain an altered gene having particular codons alteredaccording to the desired substitution, deletion, or insertion. Exemplarymethods of making the alterations set forth above are disclosed bySambrook et al. Molecular Cloning A Laboratory Manual, 2d Ed., ColdSpring Harbor Laboratory Press (1989).

Within a particularly preferred embodiment of the invention, p97 iscloned into an expression vector as a truncated cDNA with a deletion ofthe GPI anchor sequence located in the carboxy terminus of the protein.

Briefly, the p97 gene may be generated by polymerase chain reaction(PCR) using the cloned p97 cDNA as a template. The truncated p97 issynthesized using WJ47, the 5′ PCR primer encompassing coordinates 36 to60 (coordinates based on the cDNA map) and additionally containing a SnaBI restriction site. The sequence of WJ47 is 5′-GCG CTA CGT ACT CGA GGCCCC AGC CAG CCC CGA CGG CGC C-3′ (SEQ ID NO:6). The 3′ primer, WJ48,encompasses coordinates 2172 to 2193 and additionally contains both aTGA termination codon and a SnaBI restriction site. The DNA sequence ofWJ48 is 5′-CGC GTA CGT ATG ATC ATC AGC CCG AGC ACT GCT GAG ACG AC-3′(SEQ ID NO:7). Following amplification, the truncated p97 product isinserted into pNUTΔH (obtained from Palmiter (1986) PNAS 83:1261-1265)at the Sma I restriction site. The orientations of the resultingplasmids may be determined by PCR using one priming oligonucleotidewhich anneals to the vector sequence and a second primingoligonucleotide which anneals to the insert sequence. Alternatively,appropriate restriction digests can be performed to verify theorientation. Expression of the amplified sequence results in theproduction of a soluble p97 protein lacking the hydrophobic domain.

As noted above, the present invention provides recombinant expressionvectors which include either synthetic, or cDNA-derived DNA fragmentsencoding p97 or derivatives thereof, which are operably linked tosuitable transcriptional or translational regulatory elements. Suitableregulatory elements may be derived from a variety of sources, including,but not limited to, bacterial, fungal, viral, mammalian, and insectgenes. Selection of appropriate regulatory elements is dependent on thehost cell chosen, and may be readily accomplished by one of ordinaryskill in the art. Examples of regulatory elements include, inparticular, a transcriptional promoter and enhancer or RNA polymerasebinding sequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other genetic elements, such as an origin ofreplication, additional DNA restriction sites, enhancers, sequencesconferring inducibility of transcription, and selectable markers, may beincorporated into the expression vector.

DNA sequences encoding p97 may be expressed by a wide variety ofprokaryotic and eukaryotic host cells, including, but not limited to,bacterial, mammalian, yeast, fungi, viral, plant, and insect cells.Methods for transforming or transfecting such cells for expressingforeign DNA are well known in the art (see, e.g., Itakura et al., U.S.Pat. No. 4,704,362; Hinnen et al. (1978) PNAS USA 75:1929-1933; Murrayet al., U.S. Pat. No. 4,801,542; Upshall et al., U.S. Pat. No.4,935,349; Hagen et al., U.S. Pat. No. 4,784,950; Axel et al., U.S. Pat.No. 4,399,216; Goeddel et al., U.S. Pat. No. 4,766,075; and Sambrook etal., supra).

Promoters, terminators, and methods for introducing expression vectorsof an appropriate type into, for example, plant, avian, and insect cellsmay be readily accomplished by those of skill in the art. Within aparticularly preferred embodiment of the invention, p97 is expressedfrom baculoviruses (see, e.g., Luckow and Summers (1988) BioTechnology6:47; Atkinson et al. (1990) Petic. Sci. 28:215-224). The use ofbaculoviruses such as AcMNPV is particularly preferred since host insectcells express the GPI-cleaved forms of p97. p97 may be prepared fromcultures of the host/vector systems described above that express therecombinant p97. Recombinantly produced p97 may be further purified asdescribed in more detail below.

The soluble form of p97 may be prepared by culturing cells containingthe soluble p97 through the log phase of the cell's growth andcollecting the supernatant. Preferably, the supernatant is collectedprior to the time at which the cells lose viability. Soluble p97 maythen be purified as described below, in order to yield isolated solublep97. Suitable methods for purifying the soluble p97 can be selectedbased on the hydrophilic property of the soluble p97. For example, thesoluble p97 may be readily obtained by Triton X-114 Phase Separation.

In another embodiment, p97 may be isolated from cultured CHO cellsgenetically engineered to express the GPI-anchored p97. The GPI-anchoredprotein may be harvested by a brief incubation with an enzyme capable ofcleaving the GPI anchor. Such enzymes are known in the art (Ferguson(1988) Ann. Rev. Bichem. 57:285-320) and representative examples aredescribed supra. The cleaved soluble protein may be recovered from themedium, and the cells may then be returned to growth medium for furtherexpression of the protein. Cycles of growth and harvest may be repeateduntil sufficient quantities of the protein are obtained. A particularlypreferred GPI enzyme is phospholipase C (PI-PLC) which may be obtainedeither from bacterial sources (see, Low “Phospholipase Purification andQuantification” The Practical Approach Series: Cumulative Methods Index,Rickwood and Hames, eds. IRC Press, Oxford, N.Y. (1991); Kupe et al.(1989) Eur. J. Biochem. 185:151-155; Volwerk et al. (1989) J. Cell.Biochem. 39:315-325) or from recombinant sources (Koke et al. (1991)Protein Expression and Purification 2:51-58; and Henner et al. (1986)Nuc. Acids Res. 16:10383).

p97 and derivatives thereof, including the soluble p97, may be readilypurified according to the methods described herein. Briefly, p97 may bepurified either from supernatants containing solubilized p97, or fromcultured host/vector systems as described above. A variety ofpurification steps, used either alone or in combination, may be utilizedto purify p97. For example, supernatants obtained by solubilizing p97,or from host/vector cultures as described above, may be readilyconcentrated using commercially available protein concentration filters,such as an Amicon or Millipore Pellicon ultrafiltration unit, or by“salting out” the protein followed by dialysis. In addition, thesupernatants or concentrates may be applied to an affinity purificationmatrix such as an anti-p97 antibody bound to a suitable support.Alternatively, an anion exchange resin, such as a matrix or substratehaving pendant diethylaminoethyl (DEAE) groups, may be employed.Representative matrices include acrylamide, agarose, dextran, celluloseor other types commonly employed in protein purification. Similarly,cation exchangers which utilize various insoluble matrices such assulfopropyl or carboxymethyl groups may be also used.

Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps using hydrophobic RP-HPLC media, e.g.,silica gel having pendant methyl or other alipathic groups, can beemployed to further purify p97.

p97 fragments may also be generated using the techniques describedabove, with modifications well known in the art. For example, p97expression vectors may be modified so that the expressed protein is adesired fragment of p97. This protein may be isolated from theexpression system (i.e., extracted from cells), or it may be designed tobe secreted into the supernatant of the expression system, and isolatedusing techniques described above. Alternatively, full length p97 proteinmay be generated and purified, and p97 fragments may then be generatedby cleavage reactions designed to generate the desired fragment.Chemical synthesis is an alternative route to obtain the desired p97protein or fragment thereof.

In the context of the present invention, “isolated” or “purified,” asused to define the purity of p97, refer to a protein that issubstantially free of other proteins of natural or endogenous origin,and that contains less than about 5% and preferably less than about 1%by mass of protein contaminants due to the production processes. p97 maybe considered “isolated” if it is detectable as a single protein bandupon SDS-PAGE, followed by staining with Coomassie Blue.

Production of Fusion/Chimeric Proteins

The chimeric protein of the present invention can be produced using hostcells expressing a single nucleic acid encoding the entire fusionprotein or more than one nucleic acid sequence, each encoding a domainof the chimeric protein and, optionally, an amino acid or amino acidswhich will serve to link the domains. The chimeric proteins can also beproduced by chemical synthesis.

A. Host Cells

Host cells used to produce chimeric proteins are bacterial, yeast,insect, non-mammalian vertebrate, or mammalian cells; the mammaliancells include, but are not limited to, hamster, monkey, chimpanzee, dog,cat, bovine, porcine, mouse, rat, rabbit, sheep and human cells. Thehost cells can be immortalized cells (a cell line) or non-immortalized(primary or secondary) cells and can be any of a wide variety of celltypes, such as, but not limited to, fibroblasts, keratinocytes,epithelial cells (e.g., mammary epithelial cells, intestinal epithelialcells), ovary cells (e.g., Chinese hamster ovary or CHO cells),endothelial cells, glial cells, neural cells, formed elements of theblood (e.g., lymphocytes, bone marrow cells), muscle cells, hepatocytesand precursors of these somatic cell types.

Cells which contain and express DNA or RNA encoding the chimeric proteinare referred to herein as genetically modified cells. Mammalian cellswhich contain and express DNA or RNA encoding the chimeric protein arereferred to as genetically modified mammalian cells. Introduction of theDNA or RNA into cells is by a known transfection method, such aselectroporation, microinjection, microprojectile bombardment, calciumphosphate precipitation, modified calcium phosphate precipitation,cationic lipid treatment, photoporation, fusion methodologies, receptormediated transfer, or polybrene precipitation. Alternatively, the DNA orRNA can be introduced by infection with a viral vector. Methods ofproducing cells, including mammalian cells, which express DNA or RNAencoding a chimeric protein are described in co-pending patentapplications U.S. patent application Ser. No. 08/334,797, entitled “InVivo Protein Production and Delivery System for Gene Therapy”, byRichard F Selden, Douglas A. Treco and Michael W. Heartlein (filed Nov.4, 1994); U.S. patent application Ser. No. 08/334,455, entitled “In VivoProduction and Delivery of Erythropoietin or Insulinotropin for GeneTherapy”, by Richard F Selden, Douglas A. Treco and Michael W. Heartlein(filed Nov. 4, 1994) and U.S. patent application Ser. No. 08/231,439,entitled “Targeted Introduction of DNA Into Primary or Secondary Cellsand Their Use for Gene Therapy”, by Douglas A. Treco, Michael W.Heartlein and Richard F Selden (filed Apr. 20, 1994). The teachings ofeach of these applications are expressly incorporated herein byreference.

B. Nucleic Acid Constructs

A nucleic acid construct used to express the chimeric protein can be onewhich is expressed extrachromosomally (episomally) in the transfectedmammalian cell or one which integrates, either randomly or at apre-selected targeted site through homologous recombination, into therecipient cell's genome. A construct which is expressedextrachromosomally comprises, in addition to chimeric protein-encodingsequences, sequences sufficient for expression of the protein in thecells and, optionally, for replication of the construct. It typicallyincludes a promoter, chimeric protein-encoding DNA and a polyadenylationsite. The DNA encoding the chimeric protein is positioned in theconstruct in such a manner that its expression is under the control ofthe promoter. Optionally, the construct may contain additionalcomponents such as one or more of the following: a splice site, anenhancer sequence, a selectable marker gene under the control of anappropriate promoter, and an amplifiable marker gene under the controlof an appropriate promoter.

In those embodiments in which the DNA construct integrates into thecell's genome, it need include only the chimeric protein-encodingnucleic acid sequences. Optionally, it can include a promoter and anenhancer sequence, a polyadenylation site or sites, a splice site orsites, nucleic acid sequences which encode a selectable marker ormarkers, nucleic acid sequences which encode an amplifiable markerand/or DNA homologous to genomic DNA in the recipient cell to targetintegration of the DNA to a selected site in the genome (targeting DNAor DNA sequences).

C. Cell Culture Methods

Mammalian cells containing the chimeric protein-encoding DNA or RNA arecultured under conditions appropriate for growth of the cells andexpression of the DNA or RNA. Those cells which express the chimericprotein can be identified, using known methods and methods describedherein, and the chimeric protein isolated and purified, using knownmethods and methods also described herein; either with or withoutamplification of chimeric protein production. Identification can becarried out, for example, through screening genetically modifiedmammalian cells displaying a phenotype indicative of the presence of DNAor RNA encoding the chimeric protein, such as PCR screening, screeningby Southern blot analysis, or screening for the expression of thechimeric protein. Selection of cells having incorporated chimericprotein-encoding DNA may be accomplished by including a selectablemarker in the DNA construct and culturing transfected or infected cellscontaining a selectable marker gene under conditions appropriate forsurvival of only those cells which express the selectable marker gene.Further amplification of the introduced DNA construct can be effected byculturing genetically modified mammalian cells under conditionsappropriate for amplification (e.g., culturing genetically modifiedmammalian cells containing an amplifiable marker gene in the presence ofa concentration of a drug at which only cells containing multiple copiesof the amplifiable marker gene can survive).

Genetically modified mammalian cells expressing the chimeric protein canbe identified, as described herein, by detection of the expressionproduct. For example, mammalian cells expressing chimeric protein inwhich the carrier is p97 can be identified by a sandwich enzymeimmunoassay. The antibodies can be directed toward the LRP portion orthe active agent portion.

B. Preparation of Antibodies to p97

Based on the teaching of the instant specification, antibodies to mouseor human p97 have many uses including, but not limited to, the use forthe isolation and purification of p97, use in research andidentification of p97 both in vitro and in vivo, and potentialdiagnostic (e.g., monitoring conjugate dosage levels) and therapeuticuses (e.g., modulating p97-conjugate dose levels). It is, therefore,useful to briefly set forth preferred antibodies to p97, and methods ofproducing such antibodies.

Antibodies reactive against p97 are well known in the art. Additionalanti-p97 antibodies are provided by the present invention.Representative examples of anti-p97 antibodies include L235 (ATCC No. HB8466; see, Real et al. (1985) Cancer Res. 45:4401 4411; see, also, Foodet al. (1994) J. Biol. Chem. 269(4): 3034-3040), 4.1, 8.2, 96.5 and118.1 (see, Brown et al. (1981) J. Immunol. 127(2):539-546; and Brown etal. (1981) Proc. Natl. Acad. Sci. USA 78(1):539-543); and HybC (Kennardet al. (1996) Nat. Med. 2(11):1230-1235). Other monoclonal antibodies,including, but not limited to, 2C7 and 9B6, have been generated atSynapse Technologies Inc. Antibodies to the mouse p97 include, forexample, a rabbit anti-human p97 polyclonal antibody generated against afragment of the mouse p97. In the context of the present invention,antibodies are understood to include, for example, monoclonalantibodies, polyclonal antibodies, antibody fragments (e.g., Fab, andF(ab′)2) and recombinantly produced binding partners. Antibodies areunderstood to be reactive against p97 if the Ka is greater than or equalto 10⁻⁷ M.

Polyclonal antibodies may be readily generated by one of ordinary skillin the art from a variety of warm-blooded animals. Monoclonal antibodiesmay also be readily generated using conventional techniques (see, e.g.,U.S. Pat. Nos. RE 32,011, 4,902,614; 4,543,439; and 4,411,993; see,also, Kennett, McKearn, and Bechtol (eds.) Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,(1980); and Harlow and Lane (eds.) Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press (1988)). Preparation of preferredantibodies is further described in the examples section, below.

Labels

In some embodiments, the p97 conjugate is labeled to facilitate itsdetection. A “label” or a “detectable moiety” is a compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,chemical, or other physical means. For example, labels suitable for usein the present invention include, for example, radioactive labels (e.g.,32P), fluorophores (e.g., fluorescein), electron-dense reagents, enzymes(e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptensand proteins which can be made detectable, e.g., by incorporating aradiolabel into the hapten or peptide, or used to detect antibodiesspecifically reactive with the hapten or peptide.

As noted above, depending on the screening assay employed, the agent,the linker or the p97 molecule portion of a conjugate may be labeled.The particular label or detectable group used is not a critical aspectof the invention, as long as it does not significantly interfere withthe biological activity of the conjugate. The detectable group can beany material having a detectable physical or chemical property. Thus, alabel is any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.

Examples of labels suitable for use in the present invention include,but are not limited to, fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g.,³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase,alkaline phosphatase and others commonly used in an ELISA), andcolorimetric labels such as colloidal gold or colored glass or plasticbeads (e.g., polystyrene, polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. Preferably, thelabel in one embodiment is covalently bound to p97 using an isocyanatereagent for conjugating an active agent according to the invention. Inone aspect of the invention, the bifunctional isocyanate reagents of theinvention can be used to conjugate a label to p97 to form a label p97conjugate without an active agent attached thereto. The label p97conjugate may be used as an intermediate for the synthesis of a labeledconjugate according to the invention or may be used to detect the p97conjugate. As indicated above, a wide variety of labels can be used,with the choice of label depending on sensitivity required, ease ofconjugation with the desired component of the assay, stabilityrequirements, available instrumentation, and disposal provisions.Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound.

The conjugates can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymessuitable for use as labels include, but are not limited to, hydrolases,particularly phosphatases, esterases and glycosidases, or oxidotases,particularly peroxidases. Fluorescent compounds, i.e., fluorophores,suitable for use as labels include, but are not limited to, fluoresceinand its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Further examples of suitable fluorophores include,but are not limited to, eosin, TRITC-amine, quinine, fluorescein W,acridine yellow, lissamine rhodamine, B sulfonyl chloride erythroscein,ruthenium (tris, bipyridinium), Texas Red, nicotinamide adeninedinucleotide, flavin adenine dinucleotide, etc. Chemiluminescentcompounds suitable for use as labels include, but are not limited to,luciferin and 2,3-dihydrophthalazinediones, e.g., luminol. For a reviewof various labeling or signal producing systems that can be used in themethods of the present invention, see U.S. Pat. No. 4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by the use of electronic detectors such as chargecoupled devices (CCDs) or photomultipliers and the like. Similarly,enzymatic labels may be detected by providing the appropriate substratesfor the enzyme and detecting the resulting reaction product.Colorimetric or chemiluminescent labels may be detected simply byobserving the color associated with the label. Other labeling anddetection systems suitable for use in the methods of the presentinvention will be readily apparent to those of skill in the art. Suchlabeled modulators and ligands may be used in the diagnosis of a diseaseor health condition.

Pharmaceutical Compositions, and Methods of Use/Treatment/Administration

The diseases that can be treated, ameliorated or prevented using themethods of the present invention include, but are not limited to thefollowing: Mucopolysaccharidosis I (MPS I), MPS II, MPS IIIA, MPS IIIB,Metachromatic Leukodystropy (MLD), Krabbe, Pompe, CLN2, Tay-Sachs,Niemann-Pick A and B, and other lysosomal diseases. For each disease theconjugated agent would comprise a specific compound, protein or enzyme.For methods involving MPS I, the preferred compound or enzyme isα-L-iduronidase. For methods involving MPS II, the preferred compound orenzyme iduronate-2-sulfatase. For methods involving MPS IIIA, thepreferred compound or enzyme is heparan N-sulfatase. For methodsinvolving MPS IIIB the preferred compound or enzyme is α-Nacetylglucosaminidase. For methods involving Metachromatic Leukodystropy(MLD), the preferred compound or enzyme is Arylsulfatase A. For methodsinvolving Krabbe, the preferred compound or enzyme isGalactosylceramidase. For methods involving Pompe, the preferredcompound or enzyme is acid-alpha-glucosidase. For methods involving CLN,the preferred compound or enzyme is thioesterase. For methods involvingTay-Sachs, the preferred compound or enzyme is hexosaminidase A. Formethods involving Niemann-Pick A and B the preferred compound or enzymeis Acid Spingomyelinase. For methods involving other Glycogenosisdisorders the preferred compound or enzyme is glycolipidoses,mucopolysaccharidoses, oligosaccharidoses.

The p97-conjugates of the present invention can be administered with a“pharmaceutically acceptable carrier.” Such carriers encompass any ofthe standard pharmaceutical carriers, buffers and excipients, includingphosphate-buffered saline solution, water, and emulsions (such as anoil/water or water/oil emulsion), and various types of wetting agentsand/or adjuvants. Suitable pharmaceutical carriers and theirformulations are described in Remington's Pharmaceutical Sciences (MackPublishing Co., Easton, 19th ed. 1995). Preferred pharmaceuticalcarriers depend upon the intended mode of administration of the activeagent. Typical modes of administration are described below.

The term “effective amount” means a dosage sufficient to produce adesired result on a health condition, pathology, disease of a subject orfor a diagnostic purpose. The desired result may comprise a subjectiveor objective improvement in the recipient of the dosage.

A “prophylactic treatment” is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs of adisease, wherein treatment is administered for the purpose of decreasingthe risk of developing a pathology. The conjugate conjugates of theinvention may be given as a prophylactic treatment.

A “therapeutic treatment” is a treatment administered to a subject whoexhibits signs of pathology, wherein treatment is administered for thepurpose of diminishing or eliminating those pathological signs. Thesigns may be subjective or objective.

The term “composition”, as in pharmaceutical composition, is intended toencompass a product comprising the active ingredient(s), and the inertingredient(s) that make up the carrier, as well as any product whichresults, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients, or from dissociationof one or more of the ingredients, or from other types of reactions orinteractions of one or more of the ingredients. Accordingly, thepharmaceutical compositions of the present invention encompass anycomposition made by admixing a p97-agent conjugate of the presentinvention and a pharmaceutically acceptable carrier.

The term “pharmaceutical composition” indicates a composition suitablefor pharmaceutical use in a subject, including an animal or human. Apharmaceutical composition generally comprises an effective amount ofthe p97-conjugate and a pharmaceutically acceptable carrier.

The conjugates may be administered by a variety of routes. For oralpreparations, the conjugates can be used alone or in combination withappropriate additives to make tablets, powders, granules or capsules,for example, with conventional additives, such as lactose, mannitol,corn starch or potato starch; with binders, such as crystallinecellulose, cellulose derivatives, acacia, corn starch or gelatins; withdisintegrators, such as corn starch, potato starch or sodiumcarboxymethylcellulose; with lubricants, such as talc or magnesiumstearate; and if desired, with diluents, buffering agents, moisteningagents, preservatives and flavoring agents.

The p97-agent conjugates can be formulated into preparations forinjection by dissolving, suspending or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

The p97-agent conjugates can be utilized in aerosol formulation to beadministered via inhalation. The conjugates of the present invention canbe formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the p97-agent conjugates can be made into suppositories bymixing with a variety of bases such as emulsifying bases orwater-soluble bases. The conjugates of the present invention can beadministered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

Unit dosage forms of the p97-agent conjugates for oral or rectaladministration as, for instance, syrups, elixirs, and suspensions may beprovided wherein each dosage unit, for example, teaspoonful,tablespoonful, tablet or suppository, contains a predetermined amount ofthe composition containing active agent. Similarly, unit dosage formsfor injection or intravenous administration may comprise the conjugatein a composition as a solution in sterile water, normal saline oranother pharmaceutically acceptable carrier. The term “unit dosageform,” as used herein, refers to physically discrete units suitable asunitary dosages for human and animal subjects, each unit containing apredetermined quantity of conjugates of the present invention calculatedin an amount sufficient to produce the desired effect in associationwith a pharmaceutically acceptable diluent, carrier or vehicle. Thespecifications for the novel unit dosage forms of the present inventiondepend on the particular conjugate employed and the effect to beachieved, and the pharmacodynamics associated with each compound in thehost.

In practical use, the conjugates according to the invention can becombined as the active ingredient in intimate admixture with apharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier may take a wide variety of formsdepending on the form of preparation desired for administration, e.g.,oral or parenteral (including intravenous). In preparing thecompositions for oral dosage form, any of the usual pharmaceutical mediamay be employed, such as, for example, water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents and the like in thecase of oral liquid preparations, such as, for example, suspensions,elixirs and solutions; or carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents and the like in the case of oral solidpreparations such as, for example, powders, hard and soft capsules andtablets, with the solid oral preparations being preferred over theliquid preparations.

With respect to transdermal routes of administration, methods fortransdermal administration of drugs are disclosed in Remington'sPharmaceutical Sciences, 17th Edition, (Gennaro et al. Eds., MackPublishing Co., 1985). Dermal or skin patches are a preferred means fortransdermal delivery of the p97-agent conjugates of the invention.Patches preferably provide an absorption enhancer such as DMSO toincrease the absorption of the conjugates. Other methods for transdermaldrug delivery are disclosed in U.S. Pat. Nos. 5,962,012, 6,261,595, and6,261,595. Each of which is incorporated by reference in its entirety.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are commercially available. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are commercially available.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific agent, the severity of the symptoms and thesusceptibility of the subject to side effects. Preferred dosages for agiven conjugate are readily determinable by those of skill in the art bya variety of means.

In each of these aspects, the compositions include, but are not limitedto, compositions suitable for oral, rectal, topical, parenteral(including subcutaneous, intramuscular, and intravenous), pulmonary(nasal or buccal inhalation), or nasal administration, although the mostsuitable route in any given case will depend in part on the nature andseverity of the conditions being treated and on the nature of the activeingredient. Exemplary routes of administration are the oral andintravenous routes. The compositions may be conveniently presented inunit dosage form and prepared by any of the methods well-known in theart of pharmacy.

In practical use, the conjugates according to the invention can becombined as the active ingredient in intimate admixture with apharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier may take a wide variety of formsdepending on the form of preparation desired for administration, e.g.,oral or parenteral (including intravenous). In preparing thecompositions for oral dosage form, any of the usual pharmaceutical mediamay be employed, such as, for example, water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents and the like in thecase of oral liquid preparations, such as, for example, suspensions,elixirs and solutions; or carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents and the like in the case of oral solidpreparations such as, for example, powders, hard and soft capsules andtablets, with the solid oral preparations being preferred over theliquid preparations.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit form in which case solidpharmaceutical carriers are obviously employed. If desired, tablets maybe coated by standard aqueous or nonaqueous techniques. The percentageof an active agent in these compositions may, of course, be varied andmay conveniently be between about 2 percent to about 60 percent of theweight of the unit.

The conjugates of the invention are useful for therapeutic, prophylacticand diagnostic intervention in animals, and in particular in humans.

Compositions of the present invention may be administered encapsulatedin or attached to viral envelopes or vesicles. Liposomes are vesiclesformed from a bilayer membrane. Suitable vesicles include, but are notlimited to, unilamellar vesicles and multilamellar lipid vesicles orliposomes. Such vesicles and liposomes may be made from a wide range oflipid or phospholipid compounds, such as phosphatidylcholine,phosphatidic acid, phosphatidylserine, phosphatidylethanolamine,sphingomyelin, glycolipids, gangliosides, etc. using standardtechniques, such as those described in, e.g., U.S. Pat. No. 4,394,448.Such vesicles or liposomes may be used to administer conjugatesintracellularly and to deliver the conjugates to the target organs.Controlled release of a p97-composition of interest may also be achievedusing encapsulation (see, e.g., U.S. Pat. No. 5,186,941).

Any route of administration which brings the conjugates into contactwith the target cells, tissue or organ may be used. The conjugates canbe administered peripherally or centrally. The conjugates may also beadministered intravenously or by intraperitoneally. The conjugates maybe administered locally or regionally.

The dosages to be administered will depend on individual needs andcharacteristics (age, weight, severity of condition, on the desiredeffect, the active agent used, and the chosen route of administrationand treatment regimen). Preferred dosages of p97-conjugates range fromabout 0.02 pmol/kg to about 2.5 nmol/kg, and particularly preferreddosages range from 2-250 pmol/kg; alternatively, preferred doses of thep97 conjugate may be in the range of 0.02 to 2000 mg/kg. These dosageswill be influenced by the number of agent moieties associated with eachp97 molecule. In addition, dosages may be calculated based on the agentto be administered and the severity of the condition to be treated.Empirical and theoretical methods for determining dose responserelationships and optimizing the dosages employed an individual patientstherapy are well known to one of ordinary skill in the art.

The p97-conjugates of the invention are, for example, useful fortherapeutic and prophylactic intervention the treatment of lysosomalstorage diseases in animals, and in particular in humans. The subjectmethods find use in the treatment of a variety of different lysosomalstorage diseases. In certain embodiments, of particular interest is theuse of the subject methods in disease conditions where an active agenthaving desired activity has been previously identified, but in which theactive agent is not adequately targeted to the target site, area orcompartment. With such active agent, the subject methods can be used toenhance the therapeutic efficacy and therapeutic index of active agent.

Treatment is meant to encompass any beneficial outcome to a subjectassociated with administration of a conjugate including a reducedlikelihood of acquiring a disease, prevention of a disease, slowing,stopping or reversing, the progression of a disease or an ameliorationof the symptoms associated with the disease condition afflicting thehost, where amelioration or benefit is used in a broad sense to refer toat least a reduction in the severity of the disease or in a magnitude ofa parameter representative of the severity or presence of the disease,e.g., tissue damage, cell death, excess or harmful amounts of lysosomalstorage materials, symptoms, associated with the pathological conditionbeing treated, such as inflammation and pain associated therewith. Assuch, treatment also includes, but is not limited to, situations wherethe pathological condition, or at least symptoms associated therewith,are completely inhibited, e.g., prevented from happening, or stopped,e.g., terminated, such that the host no longer suffers from thepathological condition, or at least the symptoms that characterize thepathological condition.

A variety of hosts or subjects are treatable according to the subjectmethods. Generally such subjects are “mammals” or “mammalian,” wherethese terms are used broadly to describe organisms which are within theclass mammalia, including the orders carnivore (e.g., dogs and cats),rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g.,humans, chimpanzees, and monkeys). In many embodiments, the hosts orsubjects will be humans.

Methods of Screening Conjugates for Therapeutic Activity

The ability of the conjugates of the present invention to increase thedelivery of a therapeutic agent to a lysosome can be assessed in vitroby comparing the delivery of a p97 conjugated agent to a control such asthe unconjugated agent. In a preferred embodiment, the conjugate andagent are administered in vitro to cells and the localization of theconjugate within lysosomes determined. The assessment is facilitated byconjugating a label to the p97 conjugate or unconjugated agent so thatits location within the cell can be more easily determined andquantified. Methods for monitoring the localization of compounds withina cell are well known to one of ordinary skill in the art and furtherexemplified in Examples 1 and 2. Such methods are also exemplified inU.S. patent application Ser. No. 10/206,448 filed on Jul. 25, 2002 andincorporated herein by reference in its entirety.

In another functional approach, the conjugate with an agent deficient ina LSD can be contacted in vitro with cells affected by the LSD and theeffect of the conjugate on the amount of the storage material foundwithin the lysosomes compared to the effect of an equivalent amount ofthe unconjugated agent. The cell or lysosomal volumes may be measured orthe stored material directly quantitated.

In some embodiments, the invention provides a method of screening acompound for therapeutic activity in treating a lysosomal storagedisease, by contacting a cell having a lysosome with the compound,wherein the compound comprises p97 covalently linked to an enzymedeficient in a lysosomal storage disease; and then monitoring deliveryof the compound to the lysosome. The monitoring may be by means of alabel on the conjugate and detecting the label within the lysosome or bydetermining the effect of the compound on the lysosomal storage material(e.g., does it reduce the amount of storage material.) In someembodiments, the cell is human.

In a further method, clinical trials are conducted as known to one ofordinary skill in the art and has exemplified in Example 3. Morepreferably, the conjugates are administered to test animals providing ananimal model of the LSD of interest. Such animal models are well knownto one of ordinary skill in the art. See, for example, PCT PatentPublication No: WO 01/83722 which is incorporated herein by reference.

The following examples further illustrate the present invention. Theseexamples are intended merely to be illustrative of the present inventionand are not to be constructed as being limiting.

EXAMPLES Example 1: Immunocytochemical Localization of p97

The focus of our first set of experiments has been to show thatunconjugated p97 is localized to the lysosome in cultured brain cells.This is accomplished by showing “co-localization” of p97 with a markerfor lysosomes within the cell. Showing lysosomal “co-localization” isthe first step toward validating p97 as a suitable vehicle for lysosomalenzymes.

A human neuronal line was used to conduct initial experiments.Differentiated human NT2 neurons derived from a teratocarcinoma areavailable commercially from Stratagene. Human neuronal lines are themost relevant system since the p97 protein under study is of humanorigin and the ultimate target is the neural tissue within affectedpatients.

Immunocytochemical staining of p97 within cells was accomplished withthe L235 monoclonal antibody from Synapse in conjunction with afluorescein-conjugated secondary antibody to localize the L235. Theanti-p97 L235 antibody detects endogenous material (p97 is expressed innormal neuronal cells) in addition to material that has been taken upfrom the culture medium. Markers are necessary to provide anorganelle-specific fluorescence pattern against which the observed p97fluorescence pattern can be compared. Overlap of the two patternsconfirms the specific intracellular localization of p97. For thispurpose, an antibody against Cathepsin L, a lysosomal protease, was usedin combination with L235 in the immunofluorescence experiments. Thisanti-Cathepsin L antibody was raised against a C-terminal peptide ofmurine Cathepsin L and shows strong staining of lysosomes in humanneuronal cells. A Texas red-conjugated secondary antibody was used todetect the anti-Cathepsin L primary antibody. Human NT2 neurons weregrown on glass cover slips and fed for 2 hours with 0.5 mg/ml p97. Cellswere then rinsed, fixed with formaldehyde and permeabilized. Fixed cellswere co-stained with primary and secondary antibodies and mounted onslides. Cells were also treated with a stain for the nucleus, DAPI.Slides were imaged using appropriate filter sets to resolve thedifferent markers.

Experimental Methods

Cell type: Human neuronal cell lines were primarily used to conductthese experiments. Differentiated human NT2 neurons derived from ateratocarcinoma are available commercially from Stratagene. CRL10742 andCRL10442 immature cortical neurons are available from ATCC. CRL10742(designation HCN-2) was developed from a patient with Rasmussen'sencephalitis and stains for neuronal markers but not for non-neuronalmarkers. This cell line is covered by U.S. Pat. No. 5,196,315 for use inscreening methods for evaluation of chemical and biological compounds.CRL10442 (designation HCN-1A) is a brain cortical neuron derived from apatient suffering unilateral megalencephaly. Human neuronal lines arethe most relevant system to the current investigations as the p97protein under study is of human origin and the ultimate target is theneuronal tissue within affected patients. Fibroblasts from MPSI patientwere obtained from BioMarin stock of this cell line originally obtainedfrom the Coriell Cell Bank. Cells were maintained in DMEM with 10% FetalBovine Serum (FBS).

Antibodies: The mouse anti-p97 monoclonal antibody L235 was kindlyprovided by Synapse. An appropriate dilution for immunohistochemistrywas determined by titration of antibody against fixed DG44 cells untilno background signal was visible. Rabbit anti-Cathepsin L (M-19)antibody was purchased from Santa Cruz Biotech. Secondary antibodies,Donkey anti-Mouse (DAM) and Goat Anti-Rabbit (GAR) conjugated with AlexaFluor 488 or Alexa Fluor 594 were purchased from Molecular Probes andused at dilutions recommended by the manufacturer.

Fluorescent Probes: Molecular Probes Alexa Fluor protein labeling kitswere used to fluorescently tag p97 and Iduronidase. Fluorescentlylabeled Transferrin is commercially available from Molecular Probes.Lysosensor is a marker for acidic organelles commercially available fromMolecular Probes.

Equipment: Examination of fixed cells was carried out using a LeicaDMIRB with the following filter sets: Leica Filter Cube A (UV excitationrange) Excitation Filter BP340-380/Emission LP425. Leica Filter Cube 13(Blue excitation range) Excitation Filter BP450-490/Emission LP515, usedto visualize the Alexa Fluor 488 tag. Leica Filter Cube N2.1, (Greenexcitation range) Excitation Filter BP515-560/Emission LP590, used tovisualize the Alexa Fluor 594 tag.

Protein Uptake Conditions: Cells were seeded a day prior to an uptakeexperiment on coverslips within six-well plates at a density of between2 and 5e5 cells per well. Cells were washed 3 times with serum-freeDMEM+1 mg/ml BSA. Proteins for uptake were added to the cells at 60ug/ml in DMEM+1 mg/ml BSA and incubated in a 37° C. incubator with 5%CO₂ for the duration of the uptake period. Cells were then washed 3times with PBS and fixed with a commercially availableformaldehyde-based fixative available from CALTAG. Cells werepermeabilized by immersion in 70% ethanol. Antibody staining was carriedout in CALTAG permeabilization solution. All steps were separated by 3washes in PBS the first of which contained 0.1 ug/ml DAPI to stain cellnuclei. Coverslips were mounted in Molecular Probes Antifade forexamination.

Results

The immunofluorescence images depicted in FIG. 1A-1D show the results ofa co-localization experiment in human NT2 neurons using the L235 andanti Cathepsin L antibodies. The “Light Microscopy” frame of FIG. 1Ashows a single cultured human neuron observed under phase-contrast withadditional irradiation at the excitation wavelength of DAPI. Thenucleus, which is indicated by the blue fluorescent signal, is locatedin the center of the frame. The cytoplasm of this cell can be seenstreaming away from the nucleus.

The “Cathepsin Staining” frame (FIG. 1B) is the same cell viewed underirradiation with light at the excitation wavelength for Cathepsin Ldetection. The location of Cathepsin L is identified by the redfluorescence. The punctate appearance of the signal pattern seen in thisframe is characteristic of lysosomes.

The “L235 Staining” frame of (FIG. 1C) is of the same cell but nowirradiated with light at the excitation wavelength for p97 detection.The location of p97 is identified by green fluorescence. This frameshows the same type of punctate lysosomal staining pattern as can beseen in the Cathepsin L image. Careful comparison of the fluorescentpatterns in these two frames reveals that they are coincident. TheCathepsin L and the p97 are localized identically within the cell.

Confirmation of the co-localization of the p97 and Cathepsin L is shownin FIG. 1D (“Overlay”). Combining the two fluorescent signals yields aorange colored punctate pattern, a combination of the red and greenlight from the two different antibodies. These results have beenreplicated in the ATCC neuronal line CRL 10742 that is derived fromhuman brain cortical tissue.

Example 2: Intracellular Fluorescence Detection of Tagged p97

Selective tracking of endocytosed material within a background ofendogenous material requires the use of a second detection system. Forthis purpose, cells were fed with p97, which had been conjugated to thefluorescent marker Alexa Fluor 594. Observation of marker fluorescencefrom live cells permits identification of the signal derived solely fromendocytosed p97 with no contribution from endogenous material. Alysosomal marker for live cells was necessary for co-localization withthe Alexa Fluor 594 tagged p97. Such a marker is Lysosensor Green, a dyetaken up by living cells that becomes fluorescent upon exposure to theacidic environment of the late-endosomal and lysosomal compartments.Similar to the experiment outlined above, cells were fed for 2 hourswith 0.5-mg/ml p97 and washed to remove unbound material and live cellswere imaged using appropriate filter sets to resolve the differentmarkers.

Experimental Methods

See “Experimental Methods” of Example 1.

Results

The localization of endocytosed Alexa Fluor 594-p97 was determined inlive cells. Cells were observed directly on a fluorescence microscopeusing appropriate filter sets.

Selective tracking of endocytosed material within a background ofendogenous material requires the use of a second detection system. Forthis purpose, cells were fed with p97 conjugated to the fluorescentmarker Alexa Fluor 594. Observation of marker fluorescence from livecells permits identification of the signal derived solely fromendocytosed p97 with no contribution from endogenous material. Alysosomal marker for live cells was necessary for co-localization withthe Alexa Fluor 594 tagged p97. Such a marker is Lysosensor Green, a dyetaken up by living cells that becomes fluorescent upon exposure to theacidic environment of the late endosomal and lysosomal compartments.Similar to the experiment outlined above, cells were fed for 2 hourswith 0.5 mg/ml p97 and washed to remove unbound material and live cellswere imaged using appropriate filter sets to resolve the differentmarkers.

The localization of endocytosed Alexa Fluor 594-p97 was determined inlive cells. Cells were observed directly on a fluorescence microscopeusing appropriate filter sets (FIG. 2A-2C). The “Alexa Fluor 594-p97”frame (FIG. 2A) shows a living hNT neuronal cell fed with thefluorophore-tagged p97 and Lysosensor Green. The cell in this frame isobserved under irradiation at the excitation wavelength of Alexa Fluor594. The location of the endocytosed p97 is identified by the redfluorescence. The pattern is punctate and perinuclear. The “LysosensorGreen” frame (FIG. 2B) is the same cell viewed under irradiation withlight at the excitation wavelength of Lysosensor Green. The locations ofthe acidified compartments of the cell, including the lysosomes andlate-endosomes, are identified by green fluorescence. This pattern isalso punctate and perinuclear. Co-localization of the endocytosed p97and Lysosensor dye is shown in the third frame (“Overlay”) (FIG. 2C).Combining the two fluorescent signals yields an orange-colored pattern,a combination of the red and green light from the two differentfluorescent markers.

The above-experimental data (Examples 1-2) shows that p97 is localizedin the lysosomes and transported from the cell surface to the lysosomesof cultured cells. The two main transport steps required forp97-mediated delivery to brain cell lysosomes seem to occur. Synapse hasshown that the p97 molecule delivers its “payload” across the BBB. Ourresults have shown that p97 is transported to the lysosome in culturedbrain cells. Taken together these results indicate that p97 is aneffective means to deliver recombinant enzymes to LSD patients sufferingfrom neurological manifestations of the disease.

Example 3: Treatment of Patients with MPS-I Disorder

A pharmaceutical composition comprising a conjugated agent comprisinghuman α-L-iduronidase linked to p97 is prepared by methods well-known toone skilled in the art. It is preferred to administer the pharmaceuticalcomposition intravenously. The final dosage form of the fluid comprisesthe conjugated agent, normal saline, phosphate buffer at pH 5.8 andhuman albumin at 1 mg/ml. These are prepared in a bag of normal saline.

Component Composition Conjugated agent 0.05 0.5 mg/mL or 12,500-50,000units per mL (α-L-iduronidase linked to p97) Sodium chloride solution150 mM in an IV bag, 50-250 cc total volume Sodium phosphate buffer10-50 mM, pH 5.8 Human albumin 1 mg/mL

Human patients manifesting a clinical phenotype of MPS-I disorder withan α-L-iduronidase level of less than 1% of normal in leukocytes andfibroblasts are included in the study. All patients manifest someclinical evidence of visceral and soft tissue accumulation ofglycosaminoglycans with varying degrees of functional impairment.Efficacy is determined by measuring the percentage reduction in urinaryGAG excretion over time. The urinary GAG levels in MPS-I patients arecompared to normal excretion values. There is a wide range of urine GAGvalues in untreated MPS-I patients. A greater than 50% reduction inexcretion of undegraded GAGs following therapy with the conjugated agentis a valid means to measure an individual's response to therapy. Data iscollected measuring the leukocyte iduronidase activity and buccaliduronidase activity before and after therapy in MPS-I patients.Clinical assessment of liver and spleen size is performed as it is themost widely accepted means for evaluating successful bone marrowtransplant treatment in MPS-I patients (Hoogerbrugge et al., Lancet345:1398 (1995)).

Example 4: Methods and Compositions Linking Agents to the p97 Molecule

Conjugates and preferred embodiments according to the present inventioninclude those of the formula

in which A is an active agent and B is a p97 molecule for targeting ordelivery; and X₁ and X₂ are independently N or O; R is a substitutedalkyl or unsubstituted alkyl or unsubstituted or substituted heteroalkylfrom 1 to about 30 atoms in length or 1 to 50 atoms in length; and n isfrom 1 to 30. Where n is greater than 1, the active agents may be thesame or different. Where different, the active agents are useful for thetreatment of the same disease or condition. “Alkyl” encompasses divalentradicals of alkanes as defined below. Such linkers are taught in U.S.Provisional Application No. 60/395762 filed on Jul. 12, 2002 andincorporated herein by reference in its entirety.

In a further embodiment, a label, L, is covalently attached to acompound of Formula I. The label may be attached to the conjugate at theactive agent portion, the p97 portion, or the linker joining the activeagent to p97:

In some embodiments, the label is preferably attached to the p97 portionof a conjugate.

These conjugates have the advantage of release ability. When anisocyanate reagent according to the invention reacts with a hydroxygroup it forms a carbamate bond, which can be hydrolyzed by endogenousenzymes (e.g., proteases) in the body of a subject to which it isadministered. The isocyanate reagents according to the invention reactwith an amino group to generate an isourea bond, which can also behydrolyzed by endogenous enzymes in the body of a subject to which it isadministered. By virtue of attachment to the p97 moiety, the agent isdelivered to the target lysosomal compartment or site in the body, theprotease or other endogenous enzyme hydrolyzes the carbamate or isoureabond to release the free drug at the target site or compartment.

An exemplary conjugate comprises a p97 covalently linked throughfunctional group, as is well known in the art of PEGylated peptides andproteins to a PEG moiety which is in turned linked via a carbamatelinkage to the active agent. The term active agent includes, but is notlimited to, proteins or enzymes deficient in a LSD. In anotherembodiment, the conjugate is covalently linked through a carbamate groupto a PEG moiety which is in turned linked via a carbamate linkage to theactive agent. In another embodiment, the conjugate is covalently linkedthrough a carbamate group to a PEG moiety which is in turned linked viaa carbamate linkage to an alkyl or homoalkyl moiety which is in turnedlinked via a carbamate linkage to the active agent.

These conjugates also have the advantage of being synthesized with highefficiencies according to the inventive methods. The inventive reactionsbetween isocyanate groups with hydroxy and amino are very efficient; andthe yields are very high (usually over 90%). In addition, the new bondformed by the reaction of an isocyanate group with a hydroxy or an aminogroup will increase aqueous solubility of the drug. This property can beof practical importance.

Example 5: Treatment of Patients with a Lysosomal Storage DiseaseDisorder

A pharmaceutical composition comprising a conjugated agent comprising ahuman enzyme or protein deficient in a lysosomal storage disease linkedto p97 is prepared by methods well-known to one skilled in the art. Itis preferred to administer the pharmaceutical composition intravenously.Alternatively, the composition can be administered locally to theaffected organ(s). The final dosage form of the fluid comprises theconjugated agent, normal saline, phosphate buffer at pH 5.8 and humanalbumin at 1 mg/ml. These are prepared in a bag of normal saline.

Component Composition Conjugated agent 0.02 to 2.0 mg/mL (lysosomalstorage disease protein or enzyme linked to p97) Sodium chloridesolution 150 mM in an IV bag, 50-250 cc total volume Sodium phosphatebuffer 10-50 mM, pH 5.8 Human albumin 1 mg/mL

Human patients manifesting a clinical phenotype of a lysosomal storagedisease or disorder are to be treated with a conjugate having a proteinor enzyme deficient in the particular disease or disorder. All patientsmanifest some clinical evidence of excessive or harmful visceral andsoft tissue accumulation of storage material in their lysosomes asmanifested by varying degrees of functional impairment or worsenedhealth status associated with a particular lysosomal storage disease ordisorder. Preferably, enzyme levels are monitored in a patient toconfirm the absence or reduced activity of the lysosomal storage diseaseprotein in their tissues. Efficacy is determined by measuring thepercentage reduction in urinary excretion of the substrate of theconjugated enzyme over time. The urinary substrate levels in patientsare compared to normal excretion values and or levels in untreatedpatients or the same patients before treatment. Efficacy can also bedetermined according to the reduced signs and symptoms of any pathologyassociated with a lysosomal disease. Efficacy can be determined bytissue biopsy and examination of cells and or lysosomes to determine theextent by which substrate or storage material has been reduced. Efficacycan be determined by functional assessments which may be objective orsubjective (e.g., reduced pain or difficulty in function, increasedmuscle strength or stamina, increased cardiac output, exerciseendurance, changes in body mass or appearance, etc.). A greater than 25%or 50% reduction in excretion of undegraded substrate following therapywith the conjugated agent is a valid means to measure an individual'sresponse to therapy. Data may also be collected measuring the subjectconjugated enzyme's activity or presence in tissues before, after andduring therapy. Clinical assessment of organ size may be performed as ameans of assessing therapeutic efficacy (see, for instance, Hoogerbruggeet al., Lancet 345:1398 (1995)).

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention.

All publications, patents, patent applications; and web sites are hereinincorporated by reference in their entirety to the same extent as ifeach individual patent, patent application, or web site was specificallyand individually indicated to be incorporated by reference in itsentirety.

What is claimed is:
 1. A method for treating a subject having alysosomal storage disease, said method comprising administering apharmaceutical composition to the subject wherein the compositioncomprises a p97 molecule covalently linked to a protein whose deficiencycauses the disease.
 2. The method of claim 1, wherein the subject ishuman.
 3. The method of claim 1, wherein the administering isintravenous.
 4. The method of claim 1, wherein the p97 molecule is humanp97.
 5. The method of claim 1, wherein the p97 molecule is soluble p97.6. The method of claim 1, wherein the protein is α-L-iduronidase.
 7. Themethod of claim 1, wherein the p97 molecule is covalently linked to theprotein by a linker from 5 to 20 atoms in length.
 8. The method of claim1, wherein the linker is a polyethylene glycol.
 9. The method of claim1, wherein the conjugate is a fusion protein of p97 and the protein. 10.The method of claim 1, wherein the p97 molecule has as sequence which is90% identical to the sequence of a corresponding domain of human p97.11. The method of claim 1, wherein the composition comprises theconjugate in a therapeutically effective amount.
 12. The method of claim1, wherein the disease is selected from the group consisting ofaspartylglucosaminuria, cholesterol ester storage disease, Wolmandisease, cystinosis, Danon disease, Fabry disease, Farberlipogranulomatosis, Farber disease, fucosidosis, galactosialidosis typesI/II, Gaucher disease types I/II/III, Gaucher disease, globoid cellleucodystrophy, Krabbe disease, glycogen storage disease II, Pompedisease, GM1-gangliosidosis types I/II/III, GM2-gangliosidosis type I,Tay Sachs disease, GM2-gangliosidosis type II, Sandhoff disease,GM2-gangliosidosis, α-mannosidosis types I/II, β-mannosidosis,metachromatic leucodystrophy, mucolipidosis type I, sialidosis typesI/II mucolipidosis types II/III I-cell disease, mucolipidosis type IIICpseudo-Hurler polydystrophy, mucopolysaccharidosis type I,mucopolysaccharidosis type II, Hunter syndrome, mucopolysaccharidosistype IIIA, Sanfilippo syndrome, mucopolysaccharidosis type IIIB,mucopolysaccharidosis type IIIC, mucopolysaccharidosis type IIID,mucopolysaccharidosis type IVA, Morquio syndrome, mucopolysaccharidosistype IVB Morquio syndrome, mucopolysaccharidosis type VI,mucopolysaccharidosis type VII, Sly syndrome, mucopolysaccharidosis typeIX, multiple sulphatase deficiency, neuronal ceroid lipofuscinosis, CLN1Batten disease, Niemann-Pick disease types A/B, Niemann-Pick disease,Niemann-Pick disease type C1, Niemann-Pick disease type C2,pycnodysostosis, Schindler disease types I/II, Schindler disease, andsialic acid storage disease.
 13. The method of claim 1, wherein theprotein is selected from the group consisting ofaspartylglucosaminidase, acid lipase, cysteine transporter, Lamp-2,α-galactosidase A, acid ceramidase, α-L-fucosidase, β-hexosaminidase A,GM2-activator deficiency, α-D-mannosidase, β-D-mannosidase,arylsulphatase A, saposin B, neuraminidase, α-N-acetylglucosaminidasephosphotransferase, phosphotransferase γ-subunit, L-iduronidase,iduronate-2-sulphatase, heparan-N-sulphatase, α-N-acetylglucosaminidase,acetylCoA:N-acetyltransferase, N-acetylglucosamine 6-sulphatase,galactose 6-sulphatase, β-galactosidase, N-acetylgalactosamine4-sulphatase, hyaluronoglucosaminidase, multiple sulphatases, palmitoylprotein thioesterase, tripeptidyl peptidase I, acid sphingomyelinase,cholesterol trafficking, cathepsin K, α-galactosidase B, and sialic acidtransporter.
 14. A compound comprising a p97 molecule covalently linkedto a protein whose deficiency causes a lysosomal storage disease. 15.The compound of claim 14, wherein the protein is α-L-iduronidase. 16.The compound of claim 14, wherein the p97 molecule is soluble p97. 17.The compound of claim 14, wherein the compound is a fusion protein ofthe p97 molecule and the protein.
 18. The compound of claim 14, whereinthe p97 molecule is covalently linked to the protein by a linking groupwhich is 4-20 atoms in length.
 19. The compound of claim 14, wherein theconjugate is capable of passing through the blood-brain barrier andentering a lysosome of a cell within the central nervous system.
 20. Thecompound of claim 14, wherein the protein is selected from the groupconsisting of aspartylglucosaminidase, acid lipase, cysteinetransporter, Lamp-2, α-galactosidase A, acid ceramidase, α-L-fucosidase,β-hexosaminidase A, GM2-activator deficiency, α-D-mannosidase,β-D-mannosidase, arylsulphatase A, saposin B, neuraminidase,α-N-acetylglucosaminidase phosphotransferase, phosphotransferaseγ-subunit, L-iduronidase, iduronate-2-sulphatase, heparan-N-sulphatase,α-N-acetylglucosaminidase, acetylCoA:N-acetyltransferase,N-acetylglucosamine 6-sulphatase, galactose 6-sulphatase,β-galactosidase, N-acetylgalactosamine 4-sulphatase,hyaluronoglucosaminidase, multiple sulphatases, palmitoyl proteinthioesterase, tripeptidyl peptidase I, acid sphingomyelinase,cholesterol trafficking, cathepsin K, α-galactosidase B, and sialic acidtransporter.
 21. A method of screening a compound for therapeuticactivity in treating a lysosomal storage disease, said methodcomprising: contacting a cell having a lysosome with the compound,wherein the compound comprises p97 covalently linked to a proteindeficient in a lysosomal storage disease; and monitoring delivery of thecompound to the lysosome.
 22. The method of claim 21, wherein thecompound is labeled and the monitoring detects the label.
 23. The methodof claim 21, wherein the cell is human.
 24. The method of claim 21,wherein the cell is deficient in the protein.
 25. The method of claim21, wherein the monitoring is by determining the effect of the compoundon the lysosomal storage material.
 26. The method of claim 21, whereinthe cell is not protected by the blood brain barrier.
 27. Apharmaceutical composition comprising a therapeutically effective amountof compound comprising a p97 molecule covalently linked to a proteinwhose deficiency causes a lysosomal storage disease and apharmaceutically acceptable excipient.
 28. The composition of claim 27,wherein the composition is in unit dosage format.
 29. The composition ofclaim 27, wherein the protein is selected from the group consisting ofaspartylglucosaminidase, acid lipase, cysteine transporter, Lamp-2,α-galactosidase A, acid ceramidase, α-L-fucosidase, β-hexosaminidase A,GM2-activator deficiency, α-D-mannosidase, β-D-mannosidase,arylsulphatase A, saposin B, neuraminidase, α-N-acetylglucosaminidasephosphotransferase, phosphotransferase γ-subunit, L-iduronidase,iduronate-2-sulphatase, heparan-N-sulphatase, α-N-acetylglucosaminidase,acetylCoA:N-acetyltransferase, N-acetylglucosamine 6-sulphatase,galactose 6-sulphatase, β-galactosidase, N-acetylgalactosamine4-sulphatase, hyaluronoglucosaminidase, multiple sulphatases, palmitoylprotein thioesterase, tripeptidyl peptidase I, acid sphingomyelinase,cholesterol trafficking, cathepsin K, α-galactosidase B, and sialic acidtransporter.